Figure 2.
TAZ is required for expression of MES markers, invasion, self-renewal, and tumor initiation in GSC20. (A) Western analyses of basal YAP, TAZ, and CD44 levels in GSCs. (B) DNA methylation analysis by bisulfite conversion followed by sequencing of cloned PCR products. (Filled circle) Methylated CG pair; (white circle) unmethylated CG pair. Each column corresponds to 11 CpG sites proximal to the transcription start site of TAZ (shown on top). Each row represents the methylation status of a clone. (C) Real-time qPCR of TAZ, CD44, and CTGF expression in GSC8–11 after treatment with 5 μM DAC for 72 h. Fold change before and after treatment was used for comparison of transcript levels and assessed using a t-test. (D) Western analysis of TAZ, FN1, and CD44 after transient knockdown of TAZ in GSC2 and GSC20. Cells were cultured in laminin and poly-L-ornithine-coated plates and transfected with siRNA for 48 h prior to immunoblotting. (E) Western analysis of stable TAZ knockdown clones showing reduction of TAZ but not YAP levels. (F) Invasion assay of TAZ stable knockdown clones in GSC20. The invasion efficiency of the nontargeting controls was set to 100% for comparison. P-values were generated using a t-test for significance. (G) Neurosphere assay of TAZ knockdown clones. Bar graphs indicate percentage of neurosphere formation, and P-values are shown for significance. (H) Representative hematoxylin and eosin-stained brain images from SCID mice injected intracranially with the TAZ stable knockdown clones. The bottom row shows higher magnification (40×) of tumors on the top row (10×). (I) Kaplan-Meier analysis of tumor-free progression for the TAZ knockdown clones compared with the nontargeting control.